This invention relates to a method of reducing phosphate levels in a dairy stream.
A non-limiting use of sialyloligosaccharides is in infant formula. While natural human milk is widely regarded as the highly preferred source of nutrition for infants, many reasons and conditions exist that prevent mothers from feeding their infants in this manner. These reasons include abnormal levels of hormones that regulate the lactation process, such as estrogen, prolactin, and oxytocin, breast surgeries that prevent or inhibit breastfeeding, such as biopsies, lumpectomies, and mastectomies, and the presence of infectious organisms that might be passed from mother to child, including hepatitis C virus, human immunodeficiency virus, and herpes simplex viruses. Furthermore, many drugs, including those used in radiation therapy and chemotherapy for treating various cancers cannot be used in conjunction with breastfeeding for fear that they may be passed to the infant in breast milk. In addition, some mothers may choose not to breastfeed their children due to time constraints or personal choice. In these cases, synthetic infant formula is the only viable option to human milk.
Natural human milk is a complex mixture that includes primarily proteins, lipids, antibodies, and oligosaccharides. Approximately 130 oligosaccharides have been identified in human milk (McVeagh et al., 1997, J. Paediatr. Child Health, 33:281-286). The benefits of human milk are well documented and have been shown to confer immunity to infectious diseases from mother to child through secretory IgA, and to provide complete nutrition and essential glycoconjugates for nervous, digestive, and cognitive development (Gardiner, 2000, GlycoScience, 1:1-10). Oligosaccharides present in human milk have been shown to prevent cell adherence of V. cholerae, S. pneumoniae, E. coli, and H. influenzae (Holmgren et al., 1983, Infect. Immun., 39:147-154; Andersson et al., 1986, J. Infect. Dis., 153:232-237; Schwertzmann, 1999, J Pediatr. Gastroenterol. Nutr., 28:257-263; Idota et al., 1995, Biosci. Biotechnol. Biochem., 59:417-419). Of the necessary glycoconjugates in human milk, sialic acid containing oligosaccharides are some of the most prominent (Gronber et al., 1989, Carbohydr. Res. 191:261-278). Sialic acid containing glycoconjugates increase the bioavailability of vitamin B12, and are necessary for fat metabolism (Trugo, 1988, Braz. J. Med. Biol. Res., 21:883-894; Mechref et al., 1999, Glycobiology, 9:227-234). Sialyloligosaccharides have been shown to be vital components of cell membranes and membrane-bound receptors, as well as being necessary for normal brain development (Heine et al., 1993, Monatsschr. Kinderheilkd., 141:946-950). Additionally, a sialic acid supplemented diet has been correlated with increased learning behavior in mammals (Tram et al., 1997, Arch. Dis. Child., 77:315-318).
In light of the numerous previously mentioned hindrances to natural breastfeeding, and the importance of human milk to the health and development of infants, synthetic infant formulas have been developed. Although many efforts have been made to adequately reproduce all of the benefits of human milk in synthetic infant formula, long term studies have established that increased academic and cognitive abilities exist in children who were breastfed versus those who were fed infant formula (Horwood et al., 1998, Pediatrics, 101:1-7). This finding illustrates two facts. The first is that synthetic infant formulas do not yet approach parity with human milk. The second is that given the importance of sialyloligosaccharides in neurological development, infant formulas are obviously lacking when it comes to providing important sialyloligosaccharides for neurological development. Infants fed synthetic infant formula demonstrate physiological levels of sialyloligosaccharides and other sialic acid containing glycoconjugates that are 64% to 80% lower than infants fed breast milk (Sanchez-Diaz et al., 1997, J. Pediatr. Gastroenterol. Nutr., 27:405-410). Therefore, the need exists for a method of increasing the sialyloligosaccharide content of synthetic infant formulas.
Existing methods for enriching a dairy stream for sialyloligosaccharides result in relatively low yields of sialyloligosaccharides and a high phosphorous content in the product. Highly phosphorylated foodstuffs are poorly digested, can decrease the absorption of calcium and iron, complex with other divalent cations necessary for normal physiological processes, and inhibit the action of gastrointestinal enzymes. In the case of sialyloligosaccharides as a supplement to infant formula, the existing problem with foodstuffs having a high phosphate content may negate any added benefit of additional sialyloligosaccharides.
The total phosphate content of human milk ranges from approximately 150 mg/L to 240 mg/L, whereas the total phosphate content of many existing infant formulas, both those based on either bovine milk or soy milk, often exceed this range by more than two-fold. Given the previously noted problem with foods having a high phosphate content, it is apparent that there is a need to enhance the sialyloligosaccharide content of infant formula while simultaneously reducing phosphate levels. Existing methods are not capable of meeting these requirements.
A source of sialyloligosaccharides to supplement synthetic infant formula is whey, and its by-product, DeLactose Permeate (DLP). Whey is a major by-product of cheese-making which for environmental reasons, presents a difficult waste disposal problem. Whey is typically composed of about 5% by weight lactose, 1% by weight protein and about 0.5% by weight salts, where the balance of the mixture is water. DLP is produced by the ultrafiltration of whey to produce whey permeate followed by lactose crystallization resulting in DLP. While the protein component can often be recovered by ultra-filtration and accordingly used in food products, the remainder of the dairy waste stream has heretofore been of little value.
The isolation of sialyloligosaccharides is one method of increasing the economic and nutritional value of whey waste streams. Anion exchange chromatography is effective for removing charged sialyloligosaccharide components from lactose. The presence of salts, especially citrate salts from acid addition, can greatly reduce the effectiveness of sialyloligosaccharide removal such that it is conventional to remove salts from a whey waste stream in order to achieve effective recovery of sialyloligosaccharides.
Methods for removing sialyloligosaccharide fractions from a dairy stream have been reported, but the extraction and ion exchange methods have not been entirely satisfactory from the standpoint of throughput and purity. Furthermore, the low yield and high phosphorous content of sialyloligosaccharides isolated from a dairy stream thus far have made them unsuitable for use as a supplement to synthetic infant formula.
There is a long felt need for a method to isolate sialyloligosaccharides that comprises a high yield of product having a low phosphate content. The present invention meets these needs.
The invention relates to a method of processing a dairy stream comprising contacting a dairy stream with a phytase enzyme, wherein the dairy stream comprises lactose and sialyloligosaccharides.
In one embodiment, the sialyloligosaccharide is isolated after treatment of the dairy stream with a phytase.
In another embodiment, the lactose is hydrolyzed with a xcex2 galactosidase enzyme. In one aspect of this embodiment, the dairy stream is contacted by xcex2 galactosidase and phytase in the same step. In another aspect of this embodiment, the dairy stream is contacted by xcex2 galactosidase and phytase in separate steps.
The invention also relates to a method of preparing a sialyloligosaccharide-containing composition of a dairy stream, the method comprising hydrolyzing a lactose component of a dairy stream comprising lactose and a sialyloligosaccharide, contacting the dairy stream with phytase, and isolating a sialyloligosaccharide containing component, thereby preparing the sialyloligosaccharide containing component.
In one embodiment, the steps of hydrolyzing lactose, contacting the dairy stream with phytase, and isolating a sialyloligosaccharide component are performed in any order, provided that isolating the sialyloligosaccharide component is preceded by contacting the dairy stream with phytase.
In still another embodiment, the hydrolyzing of lactose is conducted with a xcex2 galactosidase enzyme.
In one embodiment of the invention, the sialyloligosaccharide component comprises a sialyloligosaccharide selected from the group consisting of 3xe2x80x2 sialyllactose, 6xe2x80x2 sialyllactose, 6xe2x80x2 sialyllactosamine, 3xe2x80x2 sialyllactosamine, disialyllactose, and a mixture thereof.
In another embodiment, the phytase is immobilized on a support.
In yet another embodiment, the phytase is in an aqueous medium.
In one embodiment of the invention, the sialyloligosaccharide component is isolated by membrane filtration. In another aspect of this embodiment, the membrane filtration is nanofiltration.
The present invention also relates to a method of producing a glucose and galactose containing composition from a dairy stream wherein the method comprises hydrolyzing a lactose component of a dairy stream comprising lactose and a sialyloligosaccharide, contacting the dairy stream with phytase, and isolating a glucose and galactose containing component, thereby preparing a glucose and galactose containing composition.
In one aspect of the present invention, the glucose and galactose containing composition comprises glucose and galactose in a ratio of about 0.95 to 1.05:1
In another aspect of the present invention, the glucose and galactose containing composition is isolated by membrane filtration.
In yet another aspect of the present invention, the membrane filtration is nanofiltration.